Staphylococcus aureus is a major cause of human pathogen that has caused a significant clinical due to acquisition of resistance to most available antibiotics. In an attempt to understand the bacterial response to cell wall-active antibiotics, nine staphylococcal proteins were identified as up-regulated employing 2-D gel electrophoresis (Singh et al., 2001). Five of these proteins were found to be homologues of an enzyme, methionine sulfoxide reductase (MsrA); signal transduction protein (TRAP); transcription elongation factor GreA; the heat shock protein GroES; and the enzyme IIA component of the PEP: sugar phosphotransferase system. We have constructed knockout mutants of the genes encoding TRAP and MsrA. Initial studies suggest that the mutations did not affect the antibiotic resistance, however, the TRAP mutant was defective in bacterial pathogenesis and the MsrA mutant showed increased sensitivity to oxidative stress. Although the role of MsrA in oxidative stress tolerance is well established, there is growing evidence suggesting the protein to be involved in multiple physiological functions. The long-term goal of this project is to study S. aureus MsrA for its roles in oxidative stress tolerance, antibiotic resistance and pathogenicity. The specific objectives of this study are: i) Molecular analysis of staphylococcal msrA genes: analysis of S. aureus genome sequence indicates the presence of a gene in the bacterial chromosome that codes for another MsrA homologue (GI: 12656489) in addition to the MsrA that we have identified to be upregulated by wall-active antibiotics. We intend to clone both the genes, overexpress them in Escherichia coli, and purify individual proteins to determine the associated methionine sulfoxide reductase activity; ii) Construction of msrA knockout mutants: both the msrA genes will be knocked out individually. Subsequently, a double msrA mutation will be constructed by the propagation of one mutation to the other; iii) Physiological studies: the msrA mutants will be tested for oxidative stress tolerance against various oxidative agents, such as H2O2, paraquat, AAPH. The impact of mutations on antibiotic resistance will also be determined; iv) Immunological studies: polyclonal antibodies against purified proteins will be raised to study the expression of the two MsrA homologues by Western blotting; v) MsrA in pathogenesis: the msrA mutants will be tested for their effect on bacterial pathogenicity using a rat model of endocarditis as described earlier (Mani et al., 1993); vi) Crystallographic studies: to identify the active site, we will determine the crystal structure of a protein encoded by a gene usually present downstream of many bacterial msrA genes. The results of the proposed studies are expected to provide detailed understanding of the MsrA in staphylococcal physiology and pathogenesis.